Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease
Activities(
)1
edition published
in
2015
in
English
and held by
1 WorldCat member
library
worldwide
Abstract: The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle
and viral interactions with their host. Here, we first describe functional characterization of ORF131b ( gp17 ) and ORF436
( gp18 ) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon
with ORF207 ( gp19 ). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from
all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding
path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases.
To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5′ → 3′
ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down
assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically
disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close
interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome
maturation and/or DNA recombination that may function in viral DNA replication/repair. Graphical abstract: Highlights: A SIRV2
operon of unknown function is conserved in archaeal linear viruses. The encoded three proteins exhibit ssDNA binding, annealing
and nuclease activities. The three proteins interact with one another. This operon may be involved in virus genome maturation
and/or DNA recombination. Contributing to a better understanding of archaeal viruses and ssDNA metabolism

Residue 146 regulates prolactin receptor folding, basal activity and ligand-responsiveness: Potential implications in breast
tumorigenesis(
)1
edition published
in
2015
in
English
and held by
1 WorldCat member
library
worldwide
Highlights: A gain-of-function mutation at position 146 disentangled in the prolactin receptor. Structural integrity was altered
in a mutation-specific manner. Structural alteration paralleled increased basal activity and ligand-insensitivity. Mutant
expression did not affect two representative breast cancer cell lines. Abstract: PRLR I146L is the first identified gain-of-function
variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations
suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used
a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based
assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that
chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the
receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it
to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7
(luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR I146D) had
minimal impact on cell proliferation and cell differentiation status